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Author Topic: Antigen Retrieval-Trypsin Method  (Read 6541 times)

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Offline ihcwor2

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Antigen Retrieval-Trypsin Method
« on: March 15, 2003, 02:49:12 AM »
Antigen Retrieval-Trypsin Method


Solutions and Reagents:

A.   10x Phosphate Buffered Saline (10xPBS):
Na2HPO4 --------------------- 10.9 g
NaH2PO4 --------------------- 3.2 g
NaCl --------------------------- 90 g
Distilled water -------------- 1000 ml
Mix to dissolve and adjust pH to 7.4

B.   1x PBS:
10xPBS -------------------------- 100 ml
Distilled water ------------------- 900 ml

C.   Trypsin Stock Solution (1%):
Trypsin --------------------------- 100 mg
             Distilled water ------------------ 10 ml
             Mix to dissolve. Aliquot and store at -20 C

D.   Calcium Chloride Stock Solution (1%):
Calcium chloride -------------- 100 mg
Distilled water ------------------ 10 ml

E.   Trypsin Working Solution (0.1%):
Trypsin stock solution (1%) ----------------- 1 ml
Calcium chloride stock solution 1%) ------ 1 ml
Distilled Water ---------------------------------- 10 ml
Adjust pH to 7.8 with 1N NaOH.


Procedure:

1.   Deparaffinize sections in 2 changes of xylene, 10 minutes each.
2.   Hydrate in absolute alcohol, 5 minutes each.
3. 95% alcohol for 5 minutes.
4. 70% alcohol for 5 minutes.
5.   Wash the sections in distilled water.
6.   Incubate in trypsin working solution for 10-20 minutes at 37 C. Time of incubation may vary depending on tissue type and degree of fixation, thus optimization may be necessary.
7.   Allow the sections to cool for 20 minutes.
8.   Rinse 2x2 min in PBS.
9.   Block endogenous enzyme activities if necessary.
10.   Wash slides in PBS.
11.   Proceed to standard immunohistochemistry staining procedure.

Antigen Retrieval-Trypsin Method
« on: March 15, 2003, 02:49:12 AM »